Figure 4.

PI4Kβ and rab11Q70L colocalize in the Golgi complex. (A) BHK-21 cells were grown on coverslips and either transfected with the indicated constructs (Myc-PI4Kβ or Myc-PI4KβkDa) or left untreated. After fixation with 4% formaldehyde, endogenous PI4Kβ was labeled with rabbit anti-PI4Kβ (PI4Kβendo), whereas the Myc-tagged proteins were labeled with rabbit anti-Myc (left, green). The cis-medial Golgi marker GM130 was labeled with mouse monoclonal antibody (middle, red). After labeling with the appropriate Cy2- and Cy3-labeled secondary antibodies the cells were examined by confocal immunofluorescence microscopy. Merged pictures are shown in the right panel. (B) BHK-21 cells were grown on coverslips and either transfected with cDNAs encoding Myc-tagged rab11wt, rab11Q70L, or rab11S25N or left untreated. After fixation with 4% formaldehyde, endogenous rab11 was labeled with rabbit antibody (rab11endo), whereas the Myc-tagged rab11 mutants were labeled with rabbit anti-Myc (left, green). The cis-medial Golgi marker GM130 was labeled with mouse monoclonal antibodies (middle, red). After labeling with the appropriate Cy2- and Cy3-labeled secondary antibodies, the cells were examined by confocal immunofluorescence microscopy. Merged pictures are shown in the right panel. (C) BHK-21 cells were grown on coverslips and either cotransfected with cDNAs encoding Myc-tagged PI4Kβ and NH-tagged rab11wt, rab11Q70L, or rab11S25N, or they were left untreated. After fixation with 4% formaldehyde, endogenous PI4Kβ (PI4Kβendo) and Myc-PI4Kβ were labeled with rabbit polyclonal antibodies (left, green), whereas endogenous rab11 (rab11endo) and NH-tagged rab11 proteins were labeled with mouse monoclonal antibodies. After labeling with the appropriate Cy2- and Cy3-labeled secondary antibodies, the cells were examined by confocal immunofluorescence microscopy. Merged pictures are shown in the right panel. Arrows indicate colocalization of PI4Kβ and rab11Q70L in vesicular structures outside the Golgi complex.